Application of bacillus velezensis and microorganism bacteria in synergistic effect

Abstract

The application discloses application of Bacillus velezensis (including artificially induced mutation or gene edited engineering bacteria) biological synergistic technology in plant carbon reduction and emission reduction green protection. By combining and synergistically applying Bacillus velezensis and at least one other microbial bacteria (including artificially induced mutation or gene edited engineering bacteria), plant disease original bacteria cells can be more efficiently destroyed, disease control efficiency can be greatly improved, soil microbial populations can be more effectively improved, and soil probiotic / pathogenic bacterial population ratio can be significantly improved; the use amount of chemical fertilizer and pesticide can be greatly reduced, carbon emission can be reduced, and even yield can be increased even under the condition that the amount of chemical fertilizer is reduced by 30%.

Description

[0001] This application is a divisional application of Chinese patent application 202510937458.8, filed on July 8, 2025, entitled "Application of Bacillus belyssus biosynergistic enhancement technology in plant carbon reduction and emission reduction green protection". Technical Field

[0002] This invention belongs to the field of agricultural microbial technology, specifically relating to the application of Bacillus belye biosynergistic enhancement technology in plant carbon reduction and emission reduction green protection. Background Technology

[0003] Bacillus belesiensis ( Bacillus velezensis Agricultural microbiology (BMS) is currently a hot research topic in the global field, particularly in green agriculture and ecological protection. BMS is known for its excellent resilience, enabling it to survive stably in complex environments. It secretes plant hormones, phosphatases, antimicrobial proteins, and lipopeptides (such as surfactants and iturobrine), exhibiting growth-promoting, phosphorus-solubilizing, and broad-spectrum antimicrobial properties. In terms of plant carbon reduction and green protection, Bacillus belye promotes plant growth, enhances photosynthetic efficiency, and improves crop carbon fixation capacity. Its biocontrol function reduces the use of chemical pesticides, lowers agricultural carbon emissions, and improves soil microbial community structure, enhancing soil carbon sequestration capacity. It plays a crucial role in achieving green and low-carbon agricultural development and has been widely applied in areas such as bio-agent development and ecological planting.

[0004] However, in practical applications, the use of Bacillus belyceae alone has certain limitations. Complex field environments (such as soil physicochemical properties and competition from other microorganisms) can easily affect its colonization and activity, leading to unstable control of soil-borne diseases. The limitations of existing screening methods also make it difficult to fully realize its efficacy. When single, inefficient, or ineffective mixed products are actually applied in the field, due to significant concentration dilution, obstacles from various environmental factors, competition from other types of live microorganisms, unfavorable competition between mixed microorganisms, and the influence of factors such as the application of chemical pesticides and fertilizers in the field, low efficacy and significant instability are easily observed.

[0005] Existing research indicates that single, inefficient, or ineffective mixtures, when applied in the field, are prone to low efficacy and significant instability due to factors such as large concentration dilution, various environmental factors, competition from other types of live microorganisms, unfavorable competition between mixed microorganisms, and the influence of chemical pesticides and fertilizers applied in the field. For example, tests conducted by the EU IRIS project in wheat fields in seven countries showed that the efficacy of a single microbial agent decreased by 92% after a 100-fold dilution (Front Plant Sci, 2021); experiments conducted by the Chinese Academy of Agricultural Sciences in acidified soil (pH < 5.0) showed that the survival rate of the strains was less than 25% (Acta Microbiologica Sinica, 2020).

[0006] Therefore, in-depth research on the scientific and effective biological synergistic combinations, mechanisms of action, and synergistic benefits between Bacillus belyssus and other microorganisms, their secondary metabolites, and plant-derived antimicrobial substances, in order to achieve a shift from single-agent antibacterial to systemic biological regulation, is a common path for agricultural microbial agents, including Bacillus belyssus. It is a stable and reliable guarantee for achieving green protection in agriculture and forestry, and a very important and necessary topic for realizing modern low-carbon agriculture and forestry. Summary of the Invention

[0007] The purpose of this invention is to provide the application of Bacillus belyssus (including naturally or artificially induced, or gene-edited engineered bacteria) biosynergistic enhancement technology in plant carbon reduction and emission reduction green protection and crop yield increase, so as to give full play to the function of Bacillus belyssus through biosynergy and promote the high-quality and sustainable development of agriculture and forestry.

[0008] The technical solution of the present invention is as follows: The fermentation broth (or powder, metabolites) of Bacillus belye is combined with at least one component B from microbial bacteria and plant-derived antimicrobial substances for the purpose of plant disease control, soil microecological restoration, and carbon reduction and yield increase. The microorganism in component B is Streptomyces ( Streptomyces ), rhizobia (Rhizobia Rhizobium Rhizobium sinense Sinorhizobium Slow-growing rhizobia Bradyrhizobium Pasteurella multocida ( ) Pasteuria penetrans arbuscular mycorrhizal fungi ( Arbuscular Mycorrhiza This includes its fermentation broth, bacterial powder, and secondary metabolites; The microbial secondary metabolites in component B include natamycin, lipopeptides (irisin, fentanyl, surfactant), and small molecule peptides (200 Da to 10 kDa). The plant-derived antibacterial substances in component B are carvacrol (or plant extracts containing carvacrol), mustard extract, and allyl isothiocyanate.

[0009] The components A (Bacillus belye) and B (Streptomyces, Rhizobium, Pasteurella multocida, Arbuscular mycorrhizal fungi) include not only natural strains but also engineered strains that have been modified from natural strains through natural or artificial mutagenesis or gene editing technologies.

[0010] The microbial secondary metabolites in component B include not only natural bacterial metabolites but also engineered bacteria derived from natural bacterial strains.

[0011] The mass ratio of Bacillus belye A to component B ranges from (100%:0) to (0:100%), excluding 0 and 100%, and consists of other excipients or newly added components besides A and B.

[0012] The synergistic effect of Bacillus belye can be applied to fields such as promoting plant growth, controlling plant diseases, soil ecological restoration, and carbon reduction and yield increase. Specific combinations include: 1. Construction of a biosynergistic control system for fungal diseases using Bacillus belyss (including naturally induced or artificially induced, or gene-edited engineered bacteria).

[0013] Option 1: Bacillus belyss fermentation broth (powder or its metabolites) A ​​+ fermentation broth (powder or its metabolites) B of other microorganisms (including naturally or artificially induced, or gene-edited engineered bacteria), wherein B is Streptomyces + Natamycin.

[0014] Option 2: Bacillus belysin fermentation broth (bacterial powder or its metabolites) A ​​+ secondary metabolites B of microorganisms (including naturally or artificially induced, or gene-edited engineered bacteria), wherein B is natamycin + lipopeptides (irisin, fenestrant, surfactant) and / or peptides (200 Da to 10 kDa).

[0015] Option 3: Bacillus vesiculosus fermentation broth (powder or its metabolites) A ​​+ plant-derived antibacterial active substance B, wherein B is carvacrol and / or carvacrol-containing plant extracts.

[0016] The Bacillus belesii biosynergistic, some of which can be co-fermented and cultured, but it is preferred to ferment them separately and then mix them in a reasonable proportion. In addition to A and B, other excipients can be added according to different purposes and requirements (the same below).

[0017] 2. Construction of a synergistic biological control system for Bacillus belyssus (including naturally induced or artificially induced, or gene-edited engineered bacteria) to prevent and control bacterial diseases.

[0018] Option 1: Bacillus vesiculosus fermentation liquid (bacterial powder or its metabolites) A ​​+ plant-derived antibacterial active substance B, wherein B is carvacrol and / or allyl isothiocyanate.

[0019] 3. Construction of a synergistic biological control system for nematode diseases using Bacillus belysus (including naturally induced or artificially induced, or gene-edited engineered bacteria).

[0020] Option 1: Bacillus vesiculosus fermentation broth (powder or its metabolites) A ​​+ fermentation broth (powder or its metabolites) B of other microorganisms (including naturally or artificially induced, or gene-edited engineered bacteria), wherein B is Pasteurella puncture.

[0021] Option 2: Bacillus vesiculosus fermentation broth (powder or its metabolites) A ​​+ plant-derived antibacterial active substance B, wherein B is mustard extract and / or allyl isothiocyanate.

[0022] 4. Construction of a biosynergistic system for carbon reduction and yield increase using Bacillus belyss (including naturally induced or artificially mutagenized or gene-edited engineered bacteria).

[0023] Option 1: Bacillus belyss fermentation broth (powder or its metabolites) A ​​+ probiotic fermentation broth (powder or its metabolites) B (including naturally or artificially induced, or gene-edited engineered bacteria), wherein B is natamycin and / or Streptomyces + slow-growing rhizobia.

[0024] Option 2: Bacillus belye fermentation broth (bacterial powder or its metabolites) A+B (lipopeptides and / or 200 Da to 10 kDa small molecule peptides + slow-growing rhizobia).

[0025] Option 3: Bacillus belye (engineered bacteria) A+B (arbuscular mycorrhizal fungi).

[0026] The application scenarios of the Bacillus belysinus biosynergistic enhancement technology solution of the present invention are as follows: Scenario 1: Soil ecological restoration and carbon emission reduction. Before planting, a synergistic combination of Bacillus belyceae (including naturally or artificially induced, or gene-edited engineered bacteria) and other microorganisms (including naturally or artificially induced, or gene-edited engineered bacteria) or biological factors, along with organic fertilizer, appropriately reduced amounts of chemical fertilizer, or other commonly used additives in production, is evenly applied to the soil tillage layer; or it is applied by watering the roots at an appropriate time. Firstly, it can effectively control target diseases in the soil, such as root rot, wilt, nematode disease, and bacterial bacterial wilt; secondly, it can effectively improve the soil microbial community. Through continuous application for 2-3 crop rotations or years, it can effectively regulate and increase the ratio of beneficial to pathogenic bacteria in the soil, even reaching above 5.0; thirdly, it regulates the soil micro-ecological environment. Beneficial microorganisms, through secreting extracellular enzymes, promote the decomposition and transformation of organic matter such as cellulose and hemicellulose in the soil, improving soil fertility, while reducing greenhouse gases such as methane produced by the anaerobic fermentation of soil organic matter, thus reducing carbon emissions in agricultural production.

[0027] Scenario 2, Seed Health Treatment. Prepare a synergistic agent of *Bacillus belyssus* (including naturally induced or artificially mutagenized, or gene-edited strains) + natamycin and / or lipopeptides and / or 200 Da–10 kDa small molecule peptides + slow-growing root nodules (including naturally induced or artificially mutagenized, or gene-edited strains) for seed dressing or seedling root treatment to prevent soil-borne diseases. Simultaneously, after *Bacillus belyssus* and other strains colonize the seedling surface, they can inhibit pathogen growth by secreting antibiotics, siderophores, and other substances, while simultaneously producing plant hormones (such as indoleacetic acid and cytokinins) to promote seed germination and root development.

[0028] Scenario 3: Green Disease Control. Compared to individual components, Bacillus belyss (including naturally induced, artificially mutagenized, or genetically edited strains) biosynergists typically exhibit a synergistic effect. In the blank control, the mycelial morphology and internal cell structure are normal. When Bacillus belyss fermentation broth is treated alone, some mycelia become swollen and deformed, branching decreases, growth is inhibited, mycelial cells become condensed, and cell membranes become irregularly wrinkled. After synergistic treatment with Bacillus belyss + natamycin + lipopeptides or peptides, the mycelia become severely deformed with a high deformity rate. The mycelia swell into gourd-shaped, spherical, or continuous clusters of spherical structures. Cellular vacuoles are very small, organelles are severely condensed, cell division does not occur, cell membranes are severely deformed or even partially dissolved, cell permeability increases significantly, and cells either do not expand or die rapidly. Attached Figure Description

[0029] Figure 1 The changes in cell structure of *Bacillus belyssus* synergistically treated with *Bacillus belyssus* compared to control (CK) are shown in the diagrams. ① shows the internal morphology of normal hyphae cells in the CK treatment; ② shows the internal morphology of hyphae cells slightly damaged by *Bacillus belyssus* alone treatment; ③ shows the internal morphology of severely damaged hyphae cells after treatment with *Bacillus belyssus* + natamycin + lipopeptide. Figure 2 A diagram illustrating the synergistic effect of Bacillus vesiculosus in promoting carbon reduction and yield increase in mung beans. Figure 2 Note: Vield Increase (production boost) Single-agent treatments Dual-bacteria Combinations Composite Treatment Full-chemical Fertilizer (CF) Amf fungi Bv Belles Rj Rhizobium Three-microbe Trimicrobial Combination Detailed Implementation

[0030] The present invention will be further described in detail below with reference to Examples 1-9.

[0031] Examples are given to illustrate the present invention, but the present invention is not limited to the following embodiments.

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0033] Example 1: Biosynergistic virulence of Bacillus belyss against Diplostomum cocovenenans I. Experiment Overview This experiment aimed to investigate the inhibitory effect and mechanism of action of *Bacillus belye* biosynthetic treatment on *Dioscorea opposita*. Multiple groups of individual and synergistic treatments were included, along with a control group (CK). The biosynergistic effect of *Bacillus belye* was evaluated using indicators such as fungal biomass; and its mechanism of action was further clarified using scanning electron microscopy and transmission electron microscopy.

[0034] II. Experimental Materials and Methods 2.1 Experimental Materials Test strain: The *Dioscorea opposita* fungus sample used in the experiment was obtained from the Plant Pathology Laboratory of China Agricultural University, and the culture medium used was PDA.

[0035] Test reagents: azoxystrobin, carvacrol, natamycin, and itursin were all purchased from the market, and their final concentration in PDA was 1:200,000; the microorganisms were isolated by our team, including Bacillus belye fermented for 72 hours and Streptomyces fermented for 120 hours, and the fermentation broth filtrate (without bacterial cells) was used.

[0036] 2.2 Experimental Methods Processing settings: The experiment included a blank control (CK) group and a positive control group containing the chemical agent azoxystrobin, with the original drug added to PDA medium at a ratio of 1:200,000.

[0037] Biological agent 1, Bacillus belye fermentation filtrate, was added to PDA medium at a volume ratio of 1:10; biological agent 2, Bacillus belye fermentation filtrate, was added to PDA medium at a volume ratio of 1:100; biological agent 3, Streptomyces fermentation filtrate, was added to PDA medium at a volume ratio of 1:100; biological agent 4, carvacrol technical grade, was added to PDA medium at a volume ratio of 1:200,000; biological agent 5, natamycin technical grade, was added to PDA medium at a volume ratio of 1:200,000; biological agent 6, iturobrine, was added to PDA medium at a volume ratio of 1:200,000.

[0038] Bacillus belyss biosynthesis 1: Bacillus belyss and Streptomyces fermentation filtrate were mixed at a 1:1 ratio, then added to PDA medium at a 1:100 volume ratio; Bacillus belyss biosynthesis 2: Carvacrol was added to Bacillus belyss fermentation filtrate at a ratio of 0.05%, then the mixture was added to PDA medium at a 1:100 volume ratio; Bacillus belyss biosynthesis 3: Natamycin was added to Bacillus belyss fermentation filtrate at a ratio of 0.05%, then the mixture was added to PDA medium at a 1:100 volume ratio; Bacillus belyss biosynthesis 4. Add 0.05% of iturin to the fermentation filtrate of Bacillus vesiculosus, and then add the mixture to PDA medium at a volume ratio of 1:100; 5. Mix Bacillus vesiculosus and Streptomyces fermentation filtrate at a 1:1 ratio, and then add it to PDA medium at a volume ratio of 1:100, followed by 0.05% natamycin; 6. Add Bacillus vesiculosus fermentation filtrate to PDA medium at a volume ratio of 1:100, and then add 0.05% natamycin and iturin (1:1).

[0039] Inoculation and culture: *Dioscorea opposita* fungus was inoculated onto infected PDA plates containing different treatment agents, with four replicates for each treatment. After inoculation, the petri dishes were placed under suitable temperature and humidity conditions for incubation.

[0040] Data processing: After culture, the growth of *Dioscorea opposita* in each replicate was measured, colony radius was recorded, and the overall mean, standard deviation, standard error, and inhibition rate were calculated for each treatment group. The inhibition rate was calculated as the percentage of growth inhibition relative to the control (CK) group.

[0041] Mechanism of action: Representative mycelia from three treatment groups—CK, Bacillus belyssin, and Bacillus belyssin + natamycin + lipopeptide—were obtained and prepared into electron microscopy sections. The internal structure of the mycelial cells was observed using transmission electron microscopy to further clarify the synergistic mechanism of action.

[0042] III. Experimental Results 3.1 Inhibitory activity.

[0043] The experimental results showed that, compared with the control and the chemical agent azoxystrobin (74.5%), the single-agent treatments, except for the 100-fold dilution of Bacillus vesiculosus (21.1%), all showed certain antibacterial effects, including Bacillus vesiculosus 10-fold dilution, Streptomyces, carvacrol, natamycin, and Iridum spp., with inhibition rates of 68.2%, 95.7%, 63.8%, 96.2%, and 97.0%, respectively (see Table 1 below).

[0044] Table 1. Survey on the synergistic inhibition of Bacillus belyssum by Diplostomum cocovenenans. ; This indicates that *Bacillus vesiculosus* exhibits superior inhibitory effects through synergistic treatment. The synergistic effects of four treatment groups—*Bacillus vesiculosus* + carvacrol, *Bacillus vesiculosus* + natamycin, *Bacillus vesiculosus* + iturobrine, and *Bacillus vesiculosus* + *Streptomyces*—were 75.1%, 91.7%, 95.8%, and 92.5%, respectively, representing increases of 54, 70.6, 74.7, and 71.4 percentage points compared to the single-agent treatment with *Bacillus vesiculosus*. Furthermore, the two ternary combination treatments—*Bacillus vesiculosus* + natamycin + *Streptomyces* and *Bacillus vesiculosus* + natamycin + iturobrine—both achieved 100% inhibition rates, demonstrating a significantly greater increase compared to the single-agent inhibition rate of *Bacillus vesiculosus*. Moreover, compared to *Bacillus vesiculosus* + natamycin, *Bacillus vesiculosus* + iturobrine, and *Bacillus vesiculosus* + *Streptomyces*, the inhibition rates were also increased by 8.3, 4.2, and 7.5 percentage points, respectively.

[0045] Meanwhile, compared with carvacrol, natamycin, iturobrine, and streptomycin alone, the inhibition rates of the four binary biological synergistic treatments increased by 11.3, 8.8, 5.9, and 11.0 percentage points, respectively.

[0046] Furthermore, the data showed that the inhibition effects of a 10-fold dilution and a 100-fold dilution of Bacillus belynsis were 68.4% and 21.6%, respectively, a difference of approximately 3.2 times. This demonstrates that the inhibitory effect of Bacillus belynsis decreases significantly with increasing dilution factor.

[0047] 3.2 Mechanism of action.

[0048] The results showed that Bacillus belye's biosynergistic effect effectively inhibited the infection and proliferation of pathogenic fungi by severely damaging the cell membrane and organelles of the pathogenic fungi, leading to mycelial malformation.

[0049] IV. Conclusion As the dilution ratio increases, the inhibitory effect of Bacillus vesiculosus decreases significantly. Especially in actual field applications, although the Bacillus vesiculosus fermentation broth itself has good antibacterial efficacy, the concentration and dosage that actually act on the pathogen are insufficient to produce good field control effects.

[0050] Bacillus vesicularis, in synergy with carvacrol, streptomycin, natamycin, and iturobacillus, exhibited a good synergistic effect, not only compared to the Bacillus vesicularis fermentation broth but also compared to the four biological elements themselves. In particular, the two ternary biological combinations demonstrated more effective antibacterial activity.

[0051] Example 2: Biosynergistic virulence of Bacillus belyssus against Ralstonia solanacearum I. Experimental Objective To investigate the inhibitory effects of Bacillus belye alone and in synergy with other biological components on Ralstonia solanacearum, and to provide a scientific basis for the prevention and control of diseases caused by Ralstonia solanacearum.

[0052] II. Experimental Materials 2.1 Test strain: Ralstonia solanacearum.

[0053] 2.2 Test reagents: copper hydroxide, Bacillus belye, allyl isothiocyanate, carvacrol.

[0054] III. Experimental Methods 3.1 Experimental Design A blank control (CK) and different antibacterial reagent treatment groups were set up, with each treatment group repeated 4 times. Antibacterial efficacy indicators were measured. Positive chemical control copper hydroxide was added to PDA medium at a ratio of 1:100,000 by weight; Belles fermentation supernatant was added to PDA medium at a ratio of 1:10 by volume; allyl isothiocyanate technical was added to PDA medium at a ratio of 1:100,000 by weight; allyl isothiocyanate was added to Belles fermentation supernatant at a ratio of 0.1% by weight, and the mixture was then added to PDA medium at a ratio of 1:10 by volume; carvacrol technical was added to PDA medium at a ratio of 1:100,000 by weight; carvacrol was added to Belles fermentation supernatant at a ratio of 0.1% by weight, and the mixture was then added to PDA medium at a ratio of 1:10 by volume.

[0055] 3.2 Determination of antibacterial effect The inhibition zone (including the diameter of the Oxford cup) was measured using Oxford cups, and the average inhibition diameter (unit: cm), standard deviation (SD), standard error (SE), and inhibition rate (%) of each treatment group were calculated.

[0056] IV. Experimental Results 4.1 Determination of antibacterial value in different treatment groups Experimental results showed that copper hydroxide had the largest average inhibition diameter, reaching 2.24 cm, with an inhibition rate of 77.7%; Bacillus belyssus + allyl isothiocyanate had an inhibition rate of 72.7%, almost comparable to chemical agents; while Bacillus belyssus + carvacrol had an inhibition rate of 50.1%.

[0057] Table 2. Survey on the synergistic inhibition of Ralstonia solanacearum by Bacillus belyi ; Furthermore, compared with Bacillus vesicularis treatment alone, the synergistic treatments of Bacillus vesicularis + allyl isothiocyanate and Bacillus vesicularis + carvacrol increased the antibacterial rate by 38.2 and 15.6 percentage points, respectively.

[0058] Table 3 Synergistic effect compared with Bacillus belysin alone ; Compared with treatments with allyl isothiocyanate and carvacrol alone, the synergistic treatments of Bacillus vesalis + allyl isothiocyanate and Bacillus vesalis + carvacrol increased the antibacterial rate by 23.6 and 36.6 percentage points, respectively.

[0059] Table 4 Synergistic effect compared with allyl isothiocyanate and carvacrol alone ; V. Experimental Conclusions Bacillus bellis exhibits some inhibitory effect against Ralstonia solanacearum, but its antibacterial efficacy is significantly lower than that of the chemical inoculant copper hydroxide when used alone. However, its antibacterial effect is significantly enhanced when synergistically combined with allyl isothiocyanate or carvacrol. This provides technical support for the practical application of using the synergistic combination of Bacillus bellis with allyl isothiocyanate or carvacrol to control diseases caused by Ralstonia solanacearum.

[0060] Example 3: Biosynergistic virulence of Bacillus belesi against root-knot nematodes I. Purpose To clarify the inhibitory effects of Bacillus belysus alone and in synergy with other microorganisms on root-knot nematodes.

[0061] II. Materials and Methods 2.1 Experimental Materials 2.1.1 Test nematode: Second instar larvae of the southern root-knot nematode.

[0062] 2.1.2 Test reagents: Fluopyram (control reagent), Bacillus vesiculosus, mustard extract, allyl isothiocyanate.

[0063] 2.2 Experimental Methods 2.2.1 Experimental Design: A blank control (CK) was set up; fluopyram positive control was prepared as a suspension at a ratio of 1:100,000; 1-fold fermentation filtrate of *Bacillus vesiculosus*; 10-fold fermentation filtrate of *Bacillus vesiculosus*; 100-fold fermentation filtrate of *Bacillus vesiculosus*; mustard extract was prepared as a suspension at a ratio of 1:100,000; mustard extract was added to 100-fold fermentation filtrate of *Bacillus vesiculosus* at a ratio of 1:100,000; allyl isothiocyanate was prepared as a suspension at a ratio of 1:100,000; allyl isothiocyanate was added to 100-fold fermentation filtrate of *Bacillus vesiculosus* at a ratio of 1:100,000.

[0064] 2.2.2 Determination of inhibitory effect: After soaking the second-instar larvae of root-knot nematodes in the pesticide solution for 24 hours, the mortality of the root-knot nematodes was observed and recorded, and the standard deviation, standard error, and lethality rate were calculated. Each treatment group had 4 replicates, and each replicate had 3 observation dishes, with 100 tested second-instar larvae placed in each dish.

[0065] III. Experimental Results The results showed that fluopyram treatment had a 100% mortality rate against nematodes; the mortality rate of Bacillus belysin fermentation filtrate varied greatly with different dilution ratios, with 1-fold, 10-fold, and 100-fold mortality rates of 97.6%, 57.3%, and 2.3%, respectively; and the mortality rates of mustard extract and allyl isothiocyanate treatments were 88.0% and 93.0%, respectively.

[0066] Table 5. Statistical table of the inhibitory effects of each treatment on second-instar larvae. ; Both the 100x fermentation filtrate of Bacillus vesiculosus with mustard extract and the 100x fermentation filtrate of Bacillus vesiculosus with allyl isothiocyanate showed a mortality rate of 100%. Compared with the 100x fermentation filtrate of Bacillus vesiculosus, the mortality rate increased by 97.7%. Compared with the two single agents, mustard extract and allyl isothiocyanate, the mortality rates increased by 12% and 7%, respectively.

[0067] IV. Conclusion Bacillus vesiculosus exhibits some inhibitory effect on nematodes, but its inhibitory effect is greatly affected by factors such as its concentration. When the fermentation broth is used alone at a dilution greater than 100 times, it is almost ineffective. However, Bacillus vesiculosus shows a significant synergistic effect when combined with mustard extract and allyl isothiocyanate, and can be applied in actual production.

[0068] Example 4: Synergistic biological control of wheat stem base rot using Bacillus belye I. Experimental Objective To clarify the field control effect of Bacillus belyi on wheat stem base rot, evaluate its advantages and disadvantages compared with chemical agents, and provide a scientific basis for practical application.

[0069] II. Materials and Methods 2.1 Test materials 2.1.1 Test crop: Wheat, in which stem rot occurred at a moderate level and the disease incidence was relatively consistent.

[0070] 2.1.2 Test reagents: Bacillus belyceae 20 billion CFU / g, carvacrol 5% aqueous solution, streptomycin, natamycin (90%); the control chemical reagent was tebuconazole suspension (430 g / L).

[0071] 2.2 Experimental Design 2.2.1 Test location: Liulihe Town, Fangshan District, Beijing. The test area covers an area of ​​2000m², of which 1500m² is for the test and the rest is for protection rows or isolation zones.

[0072] 2.2.2 Treatment Setup: A total of 7 treatments were set up, with 4 replicates per treatment, using a randomized block design with a plot area of ​​100 m². The specific treatments were as follows: Blank control (CK): 7.5 L of water; Tebuconazole: 15 mL / 667 m², diluted with water to 7.5 L; Bacillus vesiculosus: 100 g / 667 m², diluted with water to 7.5 L; Bacillus vesiculosus + Carvacrol: 7.5 g inoculum + 25 mL carvacrol, diluted with water to 7.5 L; Bacillus vesiculosus + Streptomyces: 7.5 g inoculum + 1 L Streptomyces fermentation broth, diluted with water to 7.5 L; Bacillus vesiculosus + Natamycin: 7.5 g inoculum + 3 g natamycin, diluted with water to 7.5 L; Bacillus vesiculosus + Streptomyces + Natamycin: 7.5 g inoculum + 0.5 L Streptomyces + 1.5 g natamycin, diluted with water to 7.5 L.

[0073] 2.2.3 Application time and method: Apply the pesticide to the roots in early April, before the wheat jointing stage. The spray volume is calculated at 50L / 667m², with 7.5L per plot.

[0074] III. Survey Methods 3.1 Disease Investigation: During the grain-filling stage, 5 points were randomly selected from each plot, and 50 plants were investigated at each point. The number of diseased stems and the disease severity grade were recorded. The disease severity grading criteria were as follows: Grade 0, no browning of leaf sheaths and stems; Grade 1, browning of lower leaf sheaths, no browning of stems; Grade 2, browning of the first internode at the base; Grade 3, browning of the second internode at the base, no withered white ears; Grade 4, browning of the third internode at the base and above, no withered white ears; Grade 5, withered white ears or no ears.

[0075] Calculation formula: Disease index = [∑(number of plants at this level × relative disease level value) / (total number of plants surveyed × highest level value)] × 100; Prevention and control effect (%) = [(CK disease index - treatment disease index) / CK disease index] × 100.

[0076] 3.2 Yield survey: During the harvest period, 5 points were sampled diagonally in each plot, with 50 ears of grain at each point. The yield was measured by weighing, and the average value and increase were calculated.

[0077] IV. Results and Analysis 4.1 Control effect of wheat stem base rot Table 6. Control efficacy of each treatment against wheat stem rot ; The results showed that the average control efficacy of the chemical agent tebuconazole was 76.5%, demonstrating good disease control ability. The control efficacy of *Bacillus bellis* alone was 23.1%, significantly lower than tebuconazole, but its efficacy was significantly improved when used in combination with other microorganisms. In the synergistic treatments, *Bacillus bellis* + carvacrol, *Bacillus bellis* + Streptomyces, and *Bacillus bellis* + natamycin achieved control efficacies of 78.3%, 65.4%, and 69.2%, respectively, representing increases of 55.2%, 42.3%, and 46.1% compared to *Bacillus bellis* alone. The combination of *Bacillus bellis* + natamycin + Streptomyces exhibited a more significant synergistic effect, increasing efficacy by 18.9% and 15.1% compared to *Bacillus bellis* + Streptomyces and *Bacillus bellis* + natamycin, respectively; by 6% compared to *Bacillus bellis* + carvacrol; by 61.2% compared to *Bacillus bellis* alone; and by 7.8% compared to the chemical agent tebuconazole.

[0078] 4.2 Wheat yield increase effect Table 7. Effects of each treatment on wheat yield. ; All treatments showed yield increases, with the Bacillus berreatus + natamycin + streptomycin treatment showing the largest increase (7.3%), which was 2.9 percentage points higher than tebuconazole (4.4%), 1.9% higher than Bacillus berreatus + carvacrol, 5.7% higher than Bacillus berreatus alone, and 3.5% and 3% higher than Bacillus berreatus + streptomycin and Bacillus berreatus + natamycin, respectively, demonstrating a good biological synergistic effect.

[0079] Example 5: Biosynergistic control of rice sheath blight by Bacillus belye I. Experimental Objective To clarify the effectiveness of Bacillus belyi in the field of biosynergistic control of rice sheath blight, evaluate its advantages and disadvantages, and provide a scientific basis for practical application.

[0080] II. Materials and Methods 2.1 Test materials 2.1.1 Test crop: Rice. In the field, sheath blight occurred at a moderate to severe rate and the disease incidence was relatively consistent.

[0081] 2.1.2 Test reagents: Bacillus belyceae 20 billion CFU / g, natamycin, and Streptomyces; the control chemical reagent was difenoconazole (40% aqueous solution).

[0082] 2.2 Experimental Design 2.2.1 Test site: Paddy field in Hongxin Town, Fengyang County, Anhui Province.

[0083] 2.2.2 Treatment Setup: A total of 5 treatments were set up, with 4 replicates for each treatment, using a randomized block design with a plot area of ​​100 m². The specific treatments were as follows: blank control (CK); difenoconazole, 20 ml / 667 m²; Bacillus vesiculosus, 100 g / 667 m²; Bacillus vesiculosus (50 g / 667 m²) + natamycin (10 g / 667 m²); Bacillus vesiculosus (50 g / 667 m²) + natamycin (5 g / 667 m²) + Streptomyces (1 L / 667 m² of fermentation broth).

[0084] 2.2.3 Application time and method: Apply the pesticide to the roots in July-August 2020, once every 7 days, for a total of 3 applications.

[0085] IV. Survey Methods 3.1 Disease Survey: Five random sampling points were selected from each plot, with 50 plants surveyed at each point. Disease severity was recorded. Disease severity grading criteria: Grade 0 (no disease), Grade 1 (leaf sheath disease but stem disease), Grade 3 (leaf sheath disease, invading the stem, but stem lesions covering less than half the stem), Grade 5 (stem lesions covering more than half the stem, but without lodging or breakage), Grade 7 (death and lodging). Disease index = ∑(each disease level × number of affected plants at that disease level) ( / highest grade number in the grading criteria × total number of plants surveyed) × 100; Control effect = (control group disease index - treatment group disease index) / control group disease index × 100%.

[0086] 3.2 Yield survey: During the harvest period, 5 points were sampled diagonally in each plot, with 100 ears of grain at each point. The yield was measured by weighing, and the average value and increase were calculated.

[0087] IV. Results and Analysis 4.1 Control effect on rice sheath blight The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0088] Table 8. Control efficacy of each treatment against rice sheath blight ; The average control efficacy of the chemical pesticide difenoconazole was 63.7%, while the control efficacy of Bacillus vesiculosus alone was 16.0%, significantly lower than the control pesticide. However, the efficacy was significantly improved when used in combination with other microorganisms. The control efficacy of Bacillus vesiculosus + natamycin increased to 53.6%, showing a certain biological synergistic effect. The synergistic control efficacy of Bacillus vesiculosus + natamycin + streptomycin reached 67.2%, which was not only 13.6% higher than that of Bacillus vesiculosus + natamycin, but also 3.5% higher than that of chemical pesticides, showing a more significant biological synergistic effect.

[0089] 4.2 Rice yield increase effect Table 9. Effects of each treatment on rice yield (weight of 100 spikes, kg) ; Different treatments showed varying degrees of yield increase. The combination of Bacillus vesiculosus, natamycin, and streptomycin showed the most significant synergistic effect, with a yield increase of 16.0%, higher than the 13% increase from Bacillus vesiculosus alone; higher than the 7% increase from Bacillus vesiculosus plus natamycin, further demonstrating the synergistic effect; and higher than the 6% increase from difenoconazole, indicating that high yields can still be maintained even without or with minimal use of chemical pesticides.

[0090] Example 6: Biosynergistic control of ginger wilt by Bacillus belysinus I. Experimental Objective The study aims to clarify the biosynergistic effect of Bacillus belyi in the prevention and control of ginger wilt disease, providing a scientific basis for practical application.

[0091] II. Materials and Methods 2.1 Test materials 2.1.1 Test target: Ginger wilt disease.

[0092] 2.1.2 Test reagents: Bacillus belysinus, carvacrol (5%); control chemical reagent was copper hydroxide (46% water dispersible granules).

[0093] 2.2 Experimental Design 2.2.1 Test location: Pinggu District, Beijing.

[0094] 2.2.2 Treatment Setup: Greenhouse potted plants (30cm diameter) were used, with field soil severely affected by ginger wilt being thoroughly mixed and then filled into the pots. Five treatments were set up, with each treatment replicated four times. Specific treatments were as follows: Blank control (CK); 46% copper hydroxide water-dispersible granules, 1000-fold dilution, 50mL per pot for root irrigation; Bacillus vesiculosus 48h fermentation broth, 5 billion CFU / mL, 5mL per pot diluted to 50mL for root irrigation; Bacillus vesiculosus 2.5mL + carvacrol 2mL, 50mL per pot diluted to 50mL for root irrigation; Bacillus vesiculosus 2.5mL + carvacrol 1mL + 25mL of 1000-fold diluted copper hydroxide solution (half the amount), 50mL diluted to 50mL for root irrigation.

[0095] 2.2.3 Application time: Apply when ginger is transplanted, once every 7 days, for a total of 3 applications.

[0096] III. Survey Methods Each plant was individually surveyed at harvest, and the disease severity grade was recorded. Disease severity grading criteria: Grade 0 (no disease): The plant shows no symptoms; stems, leaves, roots, and tubers are all normal. Grade 1 (mild disease): 1-2 small, water-soaked lesions appear on the tuber surface, ≤0.5 cm in diameter, not penetrating deep into the interior. Grade 3 (moderate disease): The number of lesions on the tuber increases (3-5), 0.5-1 cm in diameter; some lesions penetrate deep into the interior, and slight discoloration is visible upon cutting. Grade 5 (severe disease): Dense lesions on the tuber (>5), >1 cm in diameter; internal tissue turns brown and rots; white bacterial ooze oozes out when squeezed. Grade 7 (major disease): The entire plant withers and dies; the tuber is severely rotten, with most or all of the tissue necrotic, appearing pasty or hollow, and emitting a foul odor. Disease index = ∑ (each disease level × number of infected plants at that disease level) / (highest level in the grading standard × total number of plants surveyed) × 100; Control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0097] IV. Results and Analysis Table 10. Control efficacy of each treatment against ginger wilt disease ; The average control efficacy of the chemical agent copper hydroxide was 71.2%, while the control efficacy of Bacillus vesiculosus alone was 15.0%, significantly lower than the control agent. However, the efficacy was significantly improved when used in combination with other microorganisms. The control efficacy of Bacillus vesiculosus + carvacrol increased to 46.2%. The control efficacy of Bacillus vesiculosus + carvacrol + copper hydroxide (half the dosage) reached 71.4%. Even with half the chemical fungicide dosage, the synergistic control efficacy was still comparable to that of chemical pesticide treatment alone.

[0098] Example 7: Biosynergistic control of lettuce wilt by Bacillus belesi and its effect on soil The impact of microbial populations I. Experimental Objective To clarify the effect of Bacillus belyi in the biosynergistic control of lettuce wilt, and to explore the impact of biosynergy on soil microbial communities, so as to provide a scientific basis for practical application.

[0099] II. Materials and Methods 2.1 Test materials 2.1.1 Test target: Lettuce wilt disease.

[0100] 2.1.2 Test agents: Bacillus berreatus, natamycin, and Streptomyces; the control chemical agent was prochloraz (25% emulsifiable concentrate, 1000 times dilution for root irrigation).

[0101] 2.2 Experimental Design 2.2.1 Test location: Tongzhou District, Beijing.

[0102] 2.2.2 Treatment Setup: Greenhouse potted plants (30cm diameter) were used, with field soil from areas severely affected by lettuce wilt being thoroughly mixed and then filled into the pots. Five treatments were set up, with each treatment replicated four times. Specific treatments were as follows: Blank control (CK); 25% imazalil EC, 1000-fold dilution, 50mL per pot for root irrigation; 48h fermentation broth of Bacillus vesiculosus, 5mL per pot, diluted to 50mL with water for root irrigation; 2.5mL Bacillus vesiculosus fermentation broth + 2.5mL Streptomyces 168h fermentation broth, diluted to 50mL with water for root irrigation; 1.5mL Bacillus vesiculosus fermentation broth + 1.5mL Streptomyces vesiculosus fermentation broth + 2mL 0.1% natamycin suspension, diluted to 50mL with water for root irrigation.

[0103] 2.2.3 Application time: Apply when lettuce is transplanted, once every 7 days, for a total of 3 applications.

[0104] III. Survey Methods 3.1 Disease Survey. Each plant was surveyed individually at harvest, and the disease severity grade was recorded. Disease severity grading standards: Grade 0, no lesions in the root and stem section; Grade 1, lesions covering less than 25% of the root and stem section; Grade 3, lesions covering 25% to 50% of the root and stem section; Grade 5, lesions covering 50% to 75% of the root and stem section; Grade 7, lesions covering more than 75% of the root and stem section or the entire plant is dead. Disease index = ∑(each disease level × number of affected plants at that disease level) / (highest grade number in the grading standards × total number of plants surveyed) × 100; Control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0105] 3.2 Soil Microbial Community Survey. At lettuce harvest, the potted soil from different treatments was thoroughly mixed, coarsely sieved to remove large particles, and 100g of soil from each treatment was frozen at -20℃. The samples were immediately sent to a gene sequencing unit for high-throughput sequencing of the soil microbial community (including fungal and bacterial communities). After obtaining the sequencing results, data processing and analysis were performed.

[0106] IV. Results and Analysis 4.1 Results and analysis of the disease investigation.

[0107] Table 11. Control efficacy of various treatments against Fusarium wilt in lettuce ; Experimental results showed that the average control efficacy of the chemical agent imazalil was 70.5%, the control efficacy of Bacillus vesiculosus alone was 38.0%, and the control efficacy of Bacillus vesiculosus + Streptomyces was 53.7%. Although all were lower than the control chemical agent, they showed a certain control effect. However, the synergistic use of Bacillus vesiculosus + Streptomyces + Natamycin significantly improved the efficacy, reaching 70.3%, and the synergistic control efficacy was 16.6% higher than that of Bacillus vesiculosus + Streptomyces, and almost equivalent to that of the control chemical agent.

[0108] 4.2 Results and analysis of soil microbial population detection.

[0109] 4.2.1 Soil fungal populations.

[0110] The results showed that, at the genus level, the fungal populations in the experimental soil samples mainly comprised 19 genera, as detailed in Table 12. Analysis of the relative abundance of fungal population genomes revealed that, for the untreated control group (CK), the 10 genera with the highest soil abundance were: [List of genera that contribute to soil improvement]. Mortierella (11.17029), which may lead to leaf spot disease in plants. Alternaria (11.18747), which contributes to lignin degradation Pseudaleuria (7.812026) Helps control nematodes Arthrobotrys (5.285498), which helps in the breakdown of keratin. Aphanoascus (3.031834), or may cause wilt disease. Fusarium (1.718039), or potentially pathogenic, biocontrol, or cellulose-decomposing. Cladosporium (1.010611), or resistant to salt and alkali, and degradable to petroleum. Guehomyces (0.555836), which is conducive to low-temperature decomposition. Geomyces (0.207175), which helps with the adsorption of heavy metals. Subramaniula (0.207175).

[0111] Effects of imazalil on major fungal populations. 1) Effects on potential probiotic populations: Mortierella The abundance decreased by 83.3%, which may lead to hindered root development, weakened plant systemic resistance (ISR), and increased susceptibility to pathogens. Pseudaleuria A 94.1% decrease in abundance may lead to a slower decomposition rate of organic matter such as straw, hindering soil carbon cycling. 2) Regarding potential pathogenic fungal populations: Alternaria The abundance decreased by 25.7%, but the absolute value was still as high as 8.31% (>5% of the disease threshold), and the risk of leaf spot disease has not been eliminated; Fusarium Abundance decreased by 54.1%, but may induce drug-resistant strains; Cladosporium The abundance increased by 882%, and secondary pathogens surged, which may trigger cladoceranosis. Chaetomium An increase in abundance of 3612% may inhibit seed germination. 3) For other special populations: Aphanoascus An abundance increase of 157% indicates abnormal proliferation of keratin-decomposing bacteria, which may interfere with nitrogen balance. Acrophialophora An increase of 5320% in abundance indicates an increase in opportunistic pathogens, which may lead to increased susceptibility in plants with weakened immune systems.

[0112] The effects of Bacillus belye on major fungal populations. 1) On potential probiotic populations: Arthrobotrys Abundance increased by 286%, and nematode-preying bacteria experienced explosive growth, forming sticky bacterial webs to prey on plant-parasitic nematodes. 2) Regarding potential pathogenic bacterial populations: Alternaria The abundance decreased by 42%, significantly lower than the 5% disease risk threshold, and the risk of leaf spot disease outbreak was greatly reduced. Fusarium Abundance decreased by 9%, indicating insufficient suppression. Cladosporium Abundance decreased by 19%, remaining below the safe threshold of 0.5% to prevent secondary pathogen outbreaks. 3) For other special populations: Cephaliophora An increase of 348% in abundance helps to suppress soil-borne diseases such as Rhizoctonia solani; Guehomyces The abundance increased by 74%, which helps to improve salt and alkali tolerance. Chaetomium The abundance decreased by 81%, which helps control the risk of toxin contamination.

[0113] The effects of Bacillus belye + Streptomyces on major fungal populations. 1) Effects on potential probiotic populations: Mortierella The abundance increased by 19%, enhancing the core growth-promoting bacteria and strengthening the foundation for defense against soil-borne diseases; Acremonium An abundance increase of 695% helps activate plant disease resistance pathways. 2) Regarding potential pathogenic fungal populations: Alternaria The abundance decreased by 59%, significantly lower than the disease risk threshold (5%), demonstrating excellent control effect against leaf spot pathogens; Fusarium Abundance decreased by 20%, indicating insufficient suppression. CladosporiumA 57% decrease in abundance helps prevent secondary pathogen outbreaks. 3) For other special populations: Pseudaleuria A 20% decrease in abundance may lead to a weakening of lignin degradation capacity; Arthrobotrys A 22% decrease in abundance may lead to a weakening of the nematode's predatory function. Sagenomella An increase of 317% in abundance helps promote the carbon cycle; Penicillium A 67% decrease in abundance helps reduce the risk of toxin-producing Penicillium.

[0114] Effects of Bacillus vesiculosus + Streptomyces + Natamycin on major fungal populations. 1) Effects on potential probiotic populations: Mortierella An increase of 51.2% in abundance contributes to root growth; Arthrobotrys An increase in abundance of 332% is helpful for nematode control; Cephaliophora An increase in abundance of 234% helps control damping-off disease. 2) Regarding potential pathogenic species: Alternaria The abundance was reduced by 71.8%, significantly reducing the risk of leaf spot disease; Fusarium The abundance decreased by 41.2%, which helps control root diseases; Cladosporium The abundance decreased by 69.3%, essentially eliminating the risk of secondary pathogen outbreaks.

[0115] Table 12 Effects of each treatment on soil fungal populations ; In summary, the synergistic biological treatment of Bacillus vesicularis + Streptomyces + Natamycin, compared with the chemical control agent imazalil, as well as the biological treatments of Bacillus vesicularis and Bacillus vesicularis + Streptomyces, demonstrates significant advantages in terms of soil fungal populations, including the massive proliferation of beneficial bacteria, the multiple suppression and shrinkage of pathogenic bacteria, a more balanced ecosystem, and lower potential risks.

[0116] Compared to imazalil, Bacillus vesiculosus + Streptomyces + natamycin has: 1) An absolute advantage in probiotic proliferation, which is crucial for soil health. Mortierella The relative abundance was 16.88%, an increase of 808% compared to 1.86%; Arthrobotrys The relative abundance was 22.84%, an increase of 325% compared to 5.38%. 2) It exhibits highly effective and sustainable inhibition against pathogenic bacteria, although... Fusarium The relative abundance of 1.01% was 28% higher than that of 0.79%, but the difference was not statistically significant. Alternaria The relative abundance was 3.16%, a decrease of 62% compared to 8.31%. 3) The population's ecological balance has a fundamental advantage, with a probiotic / pathogenic bacteria value as high as 5.3, 26 times higher than 0.2, indicating that probiotics dominate the ecology; functional bacteria PseudaleuriaRetaining 3.57% is more beneficial for maintaining soil material cycling than 0.46%; in addition, biological agents have cumulative benefits over many years, while chemical pesticides also pose risks such as resistance and residues.

[0117] Compared to two biological treatments, Bacillus belysae + Streptomyces + Natamycin showed that the antibacterial spectra of Bacillus belysae and Streptomyces are complementary, effectively compensating for the weak probiotic activation of Streptomyces alone. The synergistic application of the two has a natural affinity: 1) Synergistic effect overcomes the bottleneck of single-agent treatment. Mortierella The abundance increased by 45% and 27% compared to the other two, respectively. Arthrobotrys These figures increased by 12% and 453% respectively. 2) The combined effect of multiple targeting mechanisms more effectively reduces pathogenic fungi. Alternaria The abundance decreased by 51% and 30% respectively compared to the other two. Fusarium The abundance decreased by 36% and 27%, respectively. 3) Synergistic regulation effectively balances the microecology. Compared with the two, Bacillus vesicles + Streptomyces + Natamycin also have significant advantages in key indicators. The probiotic / pathogenic ratio is as high as 5.3, which is much higher than the 1.8 of Bacillus vesicles and the 2.9 of Bacillus vesicles + Streptomyces.

[0118] 4.2.2 Soil bacterial populations.

[0119] The test results showed that the soil bacterial population in the CK (blank control) exhibited a natural distribution, with the main dominant genera including Nitrospira (3.702879), participates in nitrification and is a key functional bacterium in the soil nitrogen cycle; Blautia (1.993123), associated with organic matter decomposition, may affect soil carbon pool stability; Bacillus (0.660787), a potential probiotic with growth-promoting and disease-fighting potential.

[0120] Imazalil, a chemical agent. 1) Effects on probiotic populations. Pseudomonas The abundance increased by 1066% (from 0.488425 to 5.661823), which may enhance biocontrol capabilities in the short term, but its sustainability needs further study. Bacillus Abundance increased slightly by 14.9% (to 0.758996). 2) Impact on functional bacterial populations, Nitrospira The abundance decreased by 90.6% (to 0.349751), which may have severely inhibited nitrification, leading to a reduction in soil nitrogen availability; Flavobacterium Abundance increased by 1076% (to 0.893308), leading to opportunistic bacterial proliferation and potentially disrupting the native ecological balance. 3) Potential risks. Blautia The abundance decreased by 98.5% (to 0.028845), which may have significantly weakened the ability to decompose organic matter and affected the formation of soil structure;Agrobacterium The abundance increased by 142.4% (to 0.238876), indicating an increase in plant pathogen vectors, which may increase the risk of crown gall disease.

[0121] Bacillus belyssus, a single biological agent. 1) Significant proliferation of probiotic population. Bacillus Its abundance surged by 446% (to 3.609368), making it a dominant probiotic. Streptomyces Abundance increased by 8.1% (to 1.075546), aiding in the synthesis of secondary metabolites. 2) Functional bacterial ecological regulation. Nitrospira The abundance decreased by 90.0% (to 0.369092), and the nitrogen cycle function was still inhibited, but it was better than the imazalil treatment; Rhizobium Abundance increased by 697% (to 0.37821), significantly promoting rhizobium proliferation and enhancing nitrogen fixation capacity in legumes. 3) Potential deficiencies. Pseudomonas The abundance decreased by 77.1% (to 1.098411), which may weaken overall disease resistance; Blautia The abundance decreased by 98.2% (to 0.03555), indicating a weak ability to decompose organic matter.

[0122] Bacillus vesiculosus + Streptomyces. 1) Probiotics and functional bacteria work synergistically to enhance... Streptomyces Abundance increased by 27.3% (to 1.267252), enhancing its dominance and promoting antibiotic synthesis; Virgisporangium Its abundance increased by 87.3% (to 1.263613), which may be involved in the degradation of complex organic matter and optimize soil carbon metabolism; Bradyrhizobium Abundance increased by 70.6% (to 0.513241), slow-growing rhizobia proliferated, and the nitrogen fixation cycle was prolonged. 2) Pathogen inhibition effect. Agrobacterium The abundance decreased by 36.3% (to 0.062775), reducing the risk of plant pathogen infection. 3) Limitations, Bacillus The abundance increased by only 24.5% (to 0.922873), and the effect of promoting bacterial activation was weaker than that of Bacillus belysin alone. Nitrospira Abundance decreased by 88.5% (to 0.426347), and nitrogen cycle function did not improve significantly.

[0123] Synergistic biological treatment with Bacillus vesiculosus, Streptomyces, and Natamycin. 1) Overlapping and proliferation of probiotic populations. Bacillus The abundance reached 2.732341, which was lower than that of the single agent of Belize, but it maintained a high level in synergy with Bacillus vesiculosus + Streptomyces vesiculosus; Streptomyces Abundance 1.131329: It takes into account the dominance of the actinomycete community and forms a dual mechanism of "antibiotic + growth promotion"; KaistobacterThe abundance increased by 24.3% (to 1.343777), which may enhance the ability to degrade organic matter and adapt to stress. 2) The ecological balance of functional bacteria was optimized. Nitrospira The abundance was 0.403306, and the nitrogen cycle function was restored compared with the single agent, approaching the level of treatment with Bacillus belyssus + Streptomyces. Rhizobium An abundance of 0.342853 indicates a high abundance of nitrogen-fixing bacteria, ensuring adequate nitrogen supply to plants. 3) Integrated pathogen control. Agrobacterium The abundance decreased by 73.3% (to 0.026254), significantly reducing the risk of soil-borne diseases such as crown gall disease; Flavobacterium The abundance decreased by 63.0% (to 0.330763), inhibiting the excessive proliferation of opportunistic bacteria.

[0124] Table 13 Effects of each treatment on soil bacterial populations ; Compared to imazalil, Bacillus vesiculosus + Streptomyces + natamycin: 1) Probiotics have significant ecological advantages. Bacillus The abundance was 3.6 times that of the prochloraz treatment. Streptomyces Abundance increased by 65.6%, forming a biological control barrier; avoiding Nitrospira 1) Significant reduction in functional bacteria, maintaining basic soil nitrogen cycle function. 2) Sustainability and safety advantages: no chemical residue risk, long-term use can gradually optimize soil microecology, while prochloraz easily induces drug resistance in bacteria and pathogens.

[0125] Compared to Bacillus vesiculosus alone, the combination of Bacillus vesiculosus + Streptomyces + Natamycin offers the following advantages: 1) Increased diversity of functional bacteria. Streptomyces The abundance decreased by 13.4% compared to a single dose, but Virgisporangium The abundance of decomposing bacteria increased by 16.2%, resulting in more comprehensive ecological functions; Nitrospira Abundance increased by 9.3%, and nitrogen cycling capacity slightly improved. 2) Broad-spectrum inhibition of pathogens. Agrobacterium The abundance decreased by 54.8% compared to a single agent, resulting in more thorough control of soil-borne pathogens.

[0126] Compared to Bacillus vesiculosus + Streptomyces + Natamycin, Bacillus vesiculosus + Streptomyces has: 1) Enhanced growth-promoting bacterial advantage. Bacillus Its abundance is 2.96 times that of Bacillus belyssus + Streptomyces, and its growth-promoting effect is more prominent, which is beneficial to the development of plant roots; Rhizobium The abundance of both is similar, and their nitrogen-fixing function is not weakened. 2) The ecological balance is better, avoiding the negative impact of Bacillus belyssus + Streptomyces on nitrogen fixation. Blueberry The continuous inhibition of organic matter decomposing bacteria.

[0127] In summary, the synergistic biological treatment of Bacillus belyssus + Streptomyces + natamycin showed significant advantages in soil bacterial population regulation, enabling efficient proliferation. Bacillus Probiotics, and can also be beneficial through functional bacteria (such as...) Rhizobium , Virgisporangium The synergistic effect of these factors optimizes material cycling and simultaneously... Agrobacterium This treatment provides multiple inhibitions against potential pathogens. Compared to the chemical agent prochloraz, this synergistic treatment avoids damage to soil functional bacteria and carries no risk of resistance. Compared to single biological agents, it overcomes the limitations of single-strain action through the complementary effect of the microbial community, achieving a three-pronged regulation of "growth promotion, disease resistance, and ecological balance." In practical field applications, it can be combined with the return of organic matter to the field to further enhance the ecological niche competitive advantage of functional bacteria, gradually replacing the use of chemical pesticides and achieving sustainable development of soil health and safe crop production.

[0128] Example 8: Biosynergistic control of root-knot nematode disease in lettuce using Bacillus belye I. Purpose This study investigates the synergistic effect of Bacillus belye and other microorganisms on the control of root-knot nematode disease in lettuce, providing technical guidance for practical production.

[0129] II. Materials and Methods 2.1 Test Materials 2.1.1 Test target: root-knot nematode disease of lettuce.

[0130] 2.1.2 Test reagents: Fluopyram (control reagent), Bacillus belysin, carvacrol, Pasteurella multocida.

[0131] 2.2 Test Methods 2.2.1 Experimental Design: The experiment used greenhouse potted plants (30cm in diameter) and sandy loam field soil severely affected by root-knot nematode disease. The soil was thoroughly mixed and then filled into the pots. A total of 7 treatments were set up, with 4 replicates for each treatment. Specific treatments were as follows: Blank control (CK); Fluopyram (41.7% h content), 0.05mL / plant, diluted with water to 50mL for root irrigation; Bacillus vesicularis, 48h fermentation broth, 50mL for root irrigation; Carvacrol, 0.1% solution, 50mL for root irrigation; Pasteurella multocida, 1g of mycelial powder with a content of 1 billion CFU / g, diluted with water to 50mL for root irrigation; 25mL Bacillus vesicularis fermentation broth + 25mL Carvacrol 0.1% solution fermentation broth, mixed and then used for root irrigation; 50mL Bacillus vesicularis fermentation broth + 1g Pasteurella multocida powder with a content of 1 billion CFU / g, mixed and then used for root irrigation.

[0132] 2.2.2 Investigation of Control Effect: 60 days after transplanting, all underground roots were dug up for later use. A 10-level grading method was used to grade the root knots of the prepared roots. Grade 0: No root knots; Grade 1: Very few root knots, less than 5; Grade 2: Very few root knots, no more than 25; Grade 3: Few root knots, 26-100; Grade 4: Moderate, many root knots, but most are not connected; Grade 5: Severe, many root knots, mostly connected, root growth slightly hindered; Grade 6: Severe, very many root knots, mostly connected; Grade 7: Very severe, extensive infection, weak root growth; Grade 8: Extremely severe, extensive infection, root growth stagnant; Grade 9: Extremely severe, most or all roots rotten. Disease index = ∑(each disease level × number of infected plants at that disease level) / (highest level in the grading standard × total number of plants investigated) × 100; Control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0133] III. Results and Analysis The experimental results showed that the control efficacy of Bacillus belysin treatment was 11.6% compared to the control (CK); while compared to fluopyram's 93.26%, the control efficacy difference was as high as 81.62 percentage points, indicating that its use alone is not promising.

[0134] The combination of Bacillus belycetamol and carvacrol showed a control efficacy of 55.4% compared to the control (CK), which is still significantly lower than the 93.26% efficacy of fluopyram. However, this synergistic biotechnology improved the control efficacy by 37.8 and 13.3 percentage points, respectively, compared to the 11.6% efficacy of Bacillus belycetamol single-agent treatment and the 36.1% efficacy of carvacrol single-agent treatment.

[0135] The combination of *Bacillus belyssus* and *Pasteurella punctata* showed a control efficacy of 69.2% compared to the control (CK), which is still somewhat lower than the 93.26% efficacy of fluopyram. However, this synergistic biotechnology improved the control efficacy by 57.6 and 16.3 percentage points, respectively, compared to the 11.6% efficacy of *Bacillus belyssus* single treatment and the 52.9% efficacy of *Pasteurella punctata* single treatment.

[0136] Table 14 Statistical Table of Root Knot Disease Conditions for Each Treatment ; The results showed that *Bacillus belye*, along with carvacrol and *Pasteurella scutellariae*, exhibited significant advantages in the synergistic control of root-knot nematode disease, demonstrating a prominent synergistic effect and remarkable enhancement. This indicates a positive interaction between microorganisms, and that synergistic microbial combination is a key pathway to achieving efficient biological control. Disease control can be enhanced through complementary mechanisms (such as nutrient competition, parasitism, and antibiotic synergy). The combined efficacy of *Bacillus belye* and *Pasteurella scutellariae* reached 69.16%, showing potential to approach chemical control efficacy, demonstrating the feasibility of biological control as an alternative to chemical agents.

[0137] Example 9: Bacillus belyi synergistically promotes carbon reduction and yield increase in mung beans I. Purpose This study investigates the synergistic effects of Bacillus beleilliformis and other biological factors on mung bean yield and fertilizer usage, providing technical guidance for practical production.

[0138] II. Materials and Methods 2.1 Test Materials 2.1.1 Test crop: mung bean.

[0139] 2.1.2 Test fertilizers: compound fertilizer (control fertilizer), Bacillus belye, Japanese slow-growing rhizobium, and mycorrhizal arbuscular mycorrhizal fungi.

[0140] 2.2 Test Methods 2.2.1 Experimental Design: The experiment used greenhouse pots (30cm in diameter) and sandy loam field soil suitable for mung bean cultivation. The soil was sterilized with dry heat to kill any existing microorganisms, and then thoroughly mixed before being placed into the pots. Three mung beans were sown in each pot. After seedlings emerged, two uniformly sized seedlings were retained, and the rest were removed. A total of nine treatments were set up, with four replicates for each treatment. The specific treatments are as follows: blank control (CK); compound fertilizer (NPK, 12-18-15), 5g per pot, applied evenly to the pot before planting; Bacillus vesicularis, 48h fermentation liquid, 50mL for root irrigation; Japanese slow-growing rhizobium, 96h fermentation liquid, 50mL for root irrigation; mycorrhizal arbuscular mycorrhizal fungi, 5 billion CFU / mL, 50mL for root irrigation; Bacillus vesicularis 25mL + Japanese slow-growing rhizobium 25mL, mixed and then used for root irrigation; Bacillus vesicularis 25mL + mycorrhizal arbuscular mycorrhizal fungi 25mL, mixed and then used for root irrigation; Bacillus vesicularis 15mL + Japanese slow-growing rhizobium 20mL + mycorrhizal arbuscular mycorrhizal fungi 15mL, mixed and then used for root irrigation; Bacillus vesicularis 15mL + Japanese slow-growing rhizobium 20mL + mycorrhizal arbuscular mycorrhizal fungi 15mL + 70% compound fertilizer (3.5g), mixed and then used for root irrigation.

[0141] 2.2.2 Yield Survey: Harvesting began after the mung beans matured. Each harvested batch of mung beans was stored separately, thoroughly dried, and then weighed. Yield increase = (Treatment group weight - Control group weight) / Control group weight × 100%.

[0142] III. Results and Analysis The experimental results showed that the blank control (CK) without any treatment had a lower mung bean yield, with an average dry mung bean yield of 30.68g per pot; while the yield per pot of mung beans treated with compound fertilizer alone reached 72.64g, an increase of 136.8% compared to the CK.

[0143] Single-agent treatments with *Bacillus belye*, *Syntrophus japonicus*, and *Arbuscular mycorrhizal fungi* resulted in average yields per pot of 38.31g, 48.32g, and 35.77g, respectively, representing increases of 24.9%, 57.5%, and 16.6% compared to the control (CK), demonstrating a certain yield-increasing effect. The *Syntrophus japonicus* treatment showed particularly significant results. However, compared to compound fertilizer, the single-agent biological treatments resulted in yield reductions of 98.49%, 88.48%, and 101.03%, respectively, a substantial difference.

[0144] The synergistic treatment of Bacillus belyssus and S. nigra var. nigra resulted in an average yield of 53.66g per pot, which was 74.9% higher than the control (CK) and 61.9% lower than that of compound fertilizer alone. However, it was 50% and 17.4% higher than that of Bacillus belyssus and S. nigra var. nigra alone, respectively.

[0145] The synergistic treatment of Bacillus belyssus and mycorrhizal arbuscular fungi resulted in an average yield of 41.11g per pot, which was 34.0% higher than the control (CK) and 102.8% lower than the compound fertilizer alone. However, it was 9.1% and 17.4% higher than Bacillus belyssus and mycorrhizal arbuscular fungi alone, respectively.

[0146] Table 15. Statistics on mung bean yield for each treatment ; The average yield per pot of Bacillus belyss + Japanese slow-growing rhizobium + mycorrhizal arbuscular mycorrhizal fungi was 60.44g, which was 97.0% higher than the control (CK) and 39.8% lower than the single compound fertilizer. However, it was 22.1% and 63.0% higher than Bacillus belyss + Japanese slow-growing rhizobium and Bacillus belyss + mycorrhizal arbuscular mycorrhizal fungi, respectively.

[0147] The formula of Bacillus vesicularis + Japanese slow-growing rhizobium + mycorrhizal arbuscular mycorrhizal fungi + 70% compound fertilizer yielded an average of 82.41g per pot, which is 168.6% higher than the control (CK), 71.6% higher than the formula of Bacillus vesicularis + Japanese slow-growing rhizobium + mycorrhizal arbuscular mycorrhizal fungi, and 31.3% higher than the formula of compound fertilizer alone.

[0148] The results showed that the microbial combination exhibited excellent synergistic effects and had the potential to partially replace chemical fertilizers. The application of "Bacillus belyssus + Slow-growing rhizobium japonicum + mycorrhizal arbuscular mycorrhizal fungi + 70% compound fertilizer" achieved a 168.6% increase in mung bean yield compared to the control (CK) while reducing carbon emissions by 30%, and a 31.6% increase compared to full-volume chemical fertilizers. This demonstrates that microbial combinations are one of the optimal solutions for balancing the "carbon reduction-yield increase" contradiction, providing technical support for green agriculture.

[0149] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. The application of Bacillus belyssus and microbial strains in a synergistic effect, characterized in that, The synergistic combination of Bacillus belye and microbial strains can be used for plant disease control, soil microecological restoration, and carbon reduction and yield increase. The microorganisms are Streptomyces and Slow-growing rhizobia. Bradyrhizobium Pasteurella multocida Pasteuria penetrans Arbuscular mycorrhizal fungi Arbuscular Mycorrhiza ; The Streptomyces also includes its secondary metabolite natamycin.

2. The application of Bacillus belye and microbial strains as described in claim 1, characterized in that: The Bacillus belyssus and microorganisms mentioned include not only natural strains, but also engineered strains that have been modified from natural strains through natural or artificial mutagenesis or gene editing technologies.

3. The application of Bacillus belye and microbial strains in synergistic enhancement as described in claim 1, characterized in that, Bacillus belyssus and microbial strains include the following combinations: ① Bacillus belyssus + Pasteurella punctata; ② Bacillus belyssus + Streptomyces + Natamycin; ③ Bacillus belyssus + Slow-growing Rhizobium + Natamycin; ④ Bacillus belyssus + Streptomyces + Arbuscular Mycorrhizal Fungi; ⑤ New combinations formed by adding other components based on the above ①-④.

4. The application of Bacillus belye and microbial strains in synergistic enhancement as described in claim 1, characterized in that, The Bacillus belesii includes fermentation broth, bacterial powder, or metabolites.

5. The application of Bacillus belye and microbial strains as described in claims 1-4, characterized in that, Application scenarios include promoting plant growth, controlling plant diseases, restoring soil ecology, reducing carbon emissions and increasing yields, and other related fields.

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